Assessment of echocardiographic interpretation of dual-lumen cannula during venovenous extracorporeal membrane oxygenation use for pediatric respiratory failure.

PURPOSE: Echocardiography is considered essential during cannulation placement and manipulations. Literature evaluating transthoracic echocardiography (TTE) usage during pediatric VV-ECMO is scant. The purpose of this study is to describe the use of echocardiography during VV-ECMO at a large, quaternary children's hospital. METHODS: A retrospective, single-year cohort study was performed of pediatric patients on VV-ECMO via dual-lumen cannula at our institution from January 2019 through December 2019. For each echocardiogram, final cannula component (re-infusion port (ReP), distal tip, proximal port and distal port) positions were evaluated by one echocardiographer. For TTEs with ReP in the right atrium, two echocardiographers independently evaluated ReP direction using 2-point (Yes/No) and 4-point scales, which were semi-quantitative protocols using color Doppler images to estimate ReP jet direction to the tricuspid valve. Cohen's kappa or weighted kappa was used to measure interrater agreement. RESULTS: During study period, 11 patients (64% male) received VV-ECMO with 49 TTEs and one transesophageal echocardiogram performed. The median patient age was 4.3 years [IQR: 1.1-11.5] and median VV-ECMO run time of 192 h [90-349]. The median time between TTEs on VV-ECMO was 34 h [8.3-65]. Most common position for the ReP was the right atrium (n = 33, 67%), and ReP location was not identified in five TTEs (10%). For ReP flow direction, echocardiographers agreed on 82% of TTEs using 2-point evaluation. There was only moderate agreement between echocardiographers on the 2-point and 4-point assessments (k = .54, kw = .46 respectively). CONCLUSIONS: TTE is the predominant cardiac ultrasound modality used during VV-ECMO for pediatric respiratory failure. Subjective evaluation of VV-ECMO ReP jet direction in the right atrium is challenging, regardless of assessment method.

Feasibility of Achieving Nutritional Adequacy in Critically Ill Children with Critical Neurological Illnesses (CNIs)?-A Quaternary Hospital Experience.

The literature on the nutritional needs and outcomes of critically ill children is scarce, especially on those with critical neurological illnesses (CNIs). Current evidence shows a lower mortality in patients who achieve two-thirds of their nutritional needs during the first week of pediatric intensive care unit (PICU) admission. We hypothesized that achieving 60% of the recommended dietary intake during the first week of a PICU stay is not feasible in patients with CNI. We designed an observational retrospective cohort study where we included all index admissions to the PICU in our institution of children (1 month to 18 years) with CNI from January 2018 to June 2021. We collected patient demographics, anthropometric measures, and caloric and protein intake (enteral and parenteral) information during the first week of PICU admission. Goal adequacy for calories and protein was defined as [(intake/recommended) × 100] ≥ 60%. A total of 1112 patients were included in the nutrition assessment, 12% of whom were underweight (weight for age z score < -2). Of this group, 180 met the criteria for nutrition support evaluation. On the third day of admission, 50% of the patients < 2 years achieved caloric and protein goal adequacy, compared to 25% of patients > 2 years, with p-values of 0.0003 and 0.0004, respectively. Among the underweight patients, 60% achieved both caloric and protein goal adequacy by day 3 vs. 30% of non-underweight patients with p-values of 0.0006 and 0.002, respectively. The results show that achieving 60% of the recommended dietary intake by days 5 and 7 of admission was feasible in more than half of the patients in this cohort. Additionally, children who were evaluated by a clinical dietician during the first 48 h of PICU admission reached higher nutrition adequacy.

Identifying drivers of cost in pediatric liver transplantation.

Understanding the economics of pediatric liver transplantation (LT) is central to high-value care initiatives. We examined cost and resource utilization in pediatric LT nationally to identify drivers of cost and hospital factors associated with greater total cost of care. We reviewed 3295 children (<21 y) receiving an LT from 2010 to 2020 in the Pediatric Health Information System to study cost, both per LT and service line, and associated mortality, complications, and resource utilization. To facilitate comparisons, patients were stratified into high-cost, intermediate-cost, or low-cost tertiles based on LT cost. The median cost per LT was $150,836 [IQR $104,481-$250,129], with marked variance in cost within and between hospital tertiles. High-cost hospitals (HCHs) cared for more patients with the highest severity of illness and mortality risk levels (67% and 29%, respectively), compared to intermediate-cost (60%, 21%; p <0.001) and low-cost (51%, 16%; p <0.001) hospitals. Patients at HCHs experienced a higher prevalence of mechanical ventilation, total parental nutrition use, renal comorbidities, and surgical complications than other tertiles. Clinical (27.5%), laboratory (15.1%), and pharmacy (11.9%) service lines contributed most to the total cost. Renal comorbidities ($69,563) and total parental nutrition use ($33,192) were large, independent contributors to total cost, irrespective of the cost tertile ( p <0.001). There exists a significant variation in pediatric LT cost, with HCHs caring for more patients with higher illness acuity and resource needs. Studies are needed to examine drivers of cost and associated outcomes more granularly, with the goal of defining value and standardizing care. Such efforts may uniquely benefit the sicker patients requiring the strategic resources located within HCHs to achieve the best outcomes.

Recombinant Rod Domain of Vimentin Reduces SARS-CoV-2 Viral Replication by Blocking Spike Protein-ACE2 Interactions.

Although the SARS-CoV-2 vaccination is the primary preventive intervention, there are still few antiviral therapies available, with current drugs decreasing viral replication once the virus is intracellular. Adding novel drugs to target additional points in the viral life cycle is paramount in preventing future pandemics. The purpose of this study was to create and test a novel protein to decrease SARS-CoV-2 replication. We created the recombinant rod domain of vimentin (rhRod) in E. coli and used biolayer interferometry to measure its affinity to the SARS-CoV-2 S1S2 spike protein and the ability to block the SARS-CoV-2-ACE2 interaction. We performed plaque assays to measure rhRod's effect on SARS-CoV-2 replication in Vero E6 cells. Finally, we measured lung inflammation in SARS-CoV-2-exposed K18-hACE transgenic mice given intranasal and intraperitoneal rhRod. We found that rhRod has a high affinity for the S1S2 protein with a strong ability to block S1S2-ACE2 interactions. The daily addition of rhRod decreased viral replication in Vero E6 cells starting at 48 h at concentrations >1 µM. Finally, SARS-CoV-2-infected mice receiving rhRod had decreased lung inflammation compared to mock-treated animals. Based on our data, rhRod decreases SARS-CoV-2 replication in vitro and lung inflammation in vivo. Future studies will need to evaluate the protective effects of rhRod against additional viral variants and identify the optimal dosing scheme that both prevents viral replication and host lung injury.

Traumatic intracranial hemorrhage in pediatrics: Implications of factor XIII deficiency and consumptive coagulopathy in abusive head trauma evaluation.

For infants that present with intracranial hemorrhage in the setting of suspected abusive head trauma (AHT), the standard recommendation is to perform an evaluation for a bleeding disorder. Factor XIII (FXIII) deficiency is a rare congenital bleeding disorder associated with intracranial hemorrhages in infancy, though testing for FXIII is not commonly included in the initial hemostatic evaluation. The current pediatric literature recognizes that trauma, especially traumatic brain injury, may induce coagulopathy in children, though FXIII is often overlooked as having a role in pediatric trauma-induced coagulopathy. We report an infant that presented with suspected AHT in whom laboratory workup revealed a decreased FXIII level, which was later determined to be caused by consumption in the setting of trauma induced coagulopathy, rather than a congenital disorder. Within the Child Abuse Pediatrics Research Network (CAPNET) database, 85 out of 569 (15 %) children had FXIII testing, 3 of those tested (3.5 %) had absent FXIII activity on qualitative testing, and 2 (2.4 %) children had activity levels below 30 % on quantitative testing. In this article we review the literature on the pathophysiology and treatment of low FXIII in the setting of trauma. This case and literature review demonstrate that FXIII consumption should be considered in the setting of pediatric AHT.

Protein-templated ligand discovery via the selection of DNA-encoded dynamic libraries.

DNA-encoded chemical libraries (DELs) have become a powerful technology platform in drug discovery. Dual-pharmacophore DELs display two sets of small molecules at the termini of DNA duplexes, thereby enabling the identification of synergistic binders against biological targets, and have been successfully applied in fragment-based ligand discovery and affinity maturation of known ligands. However, dual-pharmacophore DELs identify separate binders that require subsequent linking to obtain the full ligands, which is often challenging. Here we report a protein-templated DEL selection approach that can identify full ligand/inhibitor structures from DNA-encoded dynamic libraries (DEDLs) without the need for subsequent fragment linking. Our approach is based on dynamic DNA hybridization and target-templated in situ ligand synthesis, and it incorporates and encodes the linker structures in the library, along with the building blocks, to be sampled by the target protein. To demonstrate the performance of this method, 4.35-million- and 3.00-million-member DEDLs with different library architectures were prepared, and hit selection was achieved against four therapeutically relevant target proteins.

Separation of platelets by size in a microfluidic device based on controlled incremental filtration.

The significant biological and functional differences between small and large platelets suggested by recent studies could have profound implications for transfusion medicine. However, investigating the relationship between platelet size and function is challenging because separating platelets by size without affecting their properties is difficult. A standard approach is centrifugation, but it inevitably leads to premature activation and aggregation of separated platelets. This paper describes the development and validation of a microfluidic device based on controlled incremental filtration (CIF) for separating platelets by size without the cell damage and usability limitations associated with centrifugation. Platelet samples derived from whole blood were used to evaluate the dependence of the CIF device separation performance on design parameters and flow rate, and to compare the properties of PLT fractions generated by the CIF device with those produced using a centrifugation protocol in a split-sample study. This was accomplished by quantifying the platelet size distribution, mean platelet volume (MPV), platelet-large cell ratio (P-LCR) and platelet activation before and after processing for all input and output samples. The 'large platelet' fractions produced by the CIF device and the centrifugation protocol were essentially equivalent (no significant difference in MPV and P-LCR). Platelets in the 'small platelet' fraction produced by the CIF device were significantly smaller than those produced by centrifugation (lower MPV and P-LCR). This was because the CIF 'small platelet' fraction was contaminated by much fewer large platelets (∼2-times lower recovery of >12 fL platelets) and retained the smallest platelets that were discarded by the centrifugation protocol. There was no significant difference in platelet activation between the two methods. However, centrifugation required a substantial amount of additional anticoagulant to prevent platelet aggregation during pelleting. Unlike centrifugation, the CIF device offered continuous, flow-through, single-step processing that did not cause platelet aggregation. Such a capability has the potential to accelerate the basic studies of the relationship between platelet size and function, and ultimately improve transfusion practice, particularly in the pediatric setting, where the need for low-volume, high-quality platelet transfusions is most urgent.

Estimating risk of prolonged mechanical ventilation after liver transplantation in children: PROVE-ALT score.

BACKGROUND: Children at high risk for prolonged mechanical ventilation (PMV) after liver transplantation (LT) need to be identified early to optimize pulmonary support, allocate resources, and improve surgical outcomes. We aimed to develop and validate a metric that can estimate risk for Prolonged Ventilation After LT (PROVE-ALT). METHODS: We identified preoperative risk factors for PMV by univariable analysis in a retrospective cohort of pediatric LT recipients between 2011 and 2017 (n = 205; derivation cohort). We created the PROVE-ALT score by mapping multivariable logistic regression coefficients as integers, with cutoff values using the Youden Index. We validated the score by C-statistic in a retrospectively collected separate cohort of pediatric LT recipients between 2018 and 2021 (n = 133, validation cohort). RESULTS: Among total 338 patients, 21% (n = 72) were infants; 49% (n = 167) had cirrhosis; 8% (n = 27) required continuous renal replacement therapy (CRRT); and 32% (n = 111) required management in hospital (MIH) before LT. Incidence of PMV post-LT was 20% (n = 69) and 3% (n = 12) required tracheostomy. Independent risk factors (OR [95% CI]) for PMV were cirrhosis (3.8 [1-14], p = .04); age <1-year (8.2 [2-30], p = .001); need for preoperative CRRT (6.3 [1.2-32], p = .02); and MIH before LT (12.4 [2.1-71], p = .004). PROVE-ALT score ≥8 [Range = 0-21] accurately predicted PMV in the validation cohort with 73% sensitivity and 80% specificity (AUC: 0.81; 95% CI: 0.71-0.91). CONCLUSION: PROVE-ALT can predict PMV after pediatric LT with a high degree of sensitivity and specificity. Once externally validated in other centers, PROVE-ALT will empower clinicians to plan patient-specific ventilation strategies, provide parental anticipatory guidance, and optimize hospital resources.

Pediatric Swine Model of Methicillin-Resistant Staphylococcus aureus Sepsis-Induced Coagulopathy, Disseminated Microvascular Thrombosis, and Organ Injuries.

Sepsis-induced coagulopathy leading to disseminated microvascular thrombosis is associated with high mortality and has no existing therapy. Despite the high prevalence of Gram-positive bacterial sepsis, especially methicillin-resistant Staphylococcus aureus (MRSA), there is a paucity of published Gram-positive pediatric sepsis models. Large animal models replicating sepsis-induced coagulopathy are needed to test new therapeutics before human clinical trials. HYPOTHESIS: Our objective is to develop a pediatric sepsis-induced coagulopathy swine model that last 70 hours. METHODS AND MODELS: Ten 3 weeks old piglets, implanted with telemetry devices for continuous hemodynamic monitoring, were IV injected with MRSA (n = 6) (USA300, Texas Children's Hospital 1516 strain) at 1 × 109 colony forming units/kg or saline (n = 4). Fluid resuscitation was given for heart rate greater than 50% or mean arterial blood pressure less than 30% from baseline. Acetaminophen and dextrose were provided as indicated. Point-of-care complete blood count, prothrombin time (PT), activated thromboplastin time, d-dimer, fibrinogen, and specialized coagulation assays were performed at pre- and post-injection, at 0, 24, 48, 60, and 70 hours. Piglets were euthanized and necropsies performed. RESULTS: Compared with the saline treated piglets (control), the septic piglets within 24 hours had significantly lower neurologic and respiratory scores. Over time, PT, d-dimer, and fibrinogen increased, while platelet counts and activities of factors V, VII, protein C, antithrombin, and a disintegrin and metalloproteinase with thrombospondin-1 motifs (13th member of the family) (ADAMTS-13) decreased significantly in septic piglets compared with control. Histopathologic examination showed minor focal organ injuries including microvascular thrombi and necrosis in the kidney and liver of septic piglets. INTERPRETATIONS AND CONCLUSIONS: We established a 70-hour swine model of MRSA sepsis-induced coagulopathy with signs of consumptive coagulopathy, disseminated microvascular thrombosis, and early organ injuries with histological minor focal organ injuries. This model is clinically relevant to pediatric sepsis and can be used to study dysregulated host immune response and coagulopathy to infection, identify potential early biomarkers, and to test new therapeutics.

Recent advances in microfluidic cell separation to enable centrifugation-free, low extracorporeal volume leukapheresis in pediatric patients.

Leukapheresis is a common extracorporeal procedure for leukodepletion and cellular collection. During the procedure, a patient's blood is passed through an apheresis machine to separate white blood cells (WBCs) from red blood cells (RBCs) and platelets (PLTs), which are then returned to the patient. Although it is well-tolerated by adults and older children, leukapheresis poses a significant risk to neonates and low-weight infants because the extracorporeal volume (ECV) of a typical leukapheresis circuit represents a particularly large fraction of their total blood volume. The reliance of existing apheresis technology on centrifugation for separating blood cells limits the degree to which the circuit ECV could be miniaturized. The rapidly advancing field of microfluidic cell separation holds excellent promise for devices with competitive separation performance and void volumes that are orders of magnitude smaller than their centrifugation-based counterparts. This review discusses recent advancements in the field, focusing on passive separation methods that could potentially be adapted to perform leukapheresis. We first outline the performance requirements that any separation method must meet to replace centrifugation-based methods successfully. We then provide an overview of the passive separation methods that can remove WBCs from whole blood, focusing on the technological advancements made in the last decade. We describe and compare standard performance metrics, including blood dilution requirements, WBC separation efficiency, RBC and PLT loss, and processing throughput, and discuss the potential of each separation method for future use as a high-throughput microfluidic leukapheresis platform. Finally, we outline the primary common challenges that must still be overcome for these novel microfluidic technologies to enable centrifugation-free, low-ECV leukapheresis in the pediatric setting.

Elevated bile acids are associated with left ventricular structural changes in biliary atresia.

BACKGROUND: In children with biliary atresia (BA), pathologic structural changes within the heart, which define cirrhotic cardiomyopathy, are associated with adverse perioperative outcomes. Despite their clinical relevance, little is known about the pathogenesis and triggers of pathologic remodeling. Bile acid excess causes cardiomyopathy in experimental cirrhosis, but its role in BA is poorly understood. METHODS: Echocardiographic parameters of left ventricular (LV) geometry [LV mass (LVM), LVM indexed to height, left atrial volume indexed to BSA (LAVI), and LV internal diameter (LVID)] were correlated with circulating serum bile acid concentrations in 40 children (52% female) with BA listed for transplantation. A receiver-operating characteristic curve was generated to determine optimal threshold values of bile acids to detect pathologic changes in LV geometry using Youden index. Paraffin-embedded human heart tissue was separately analyzed by immunohistochemistry for the presence of bile acid-sensing Takeda G-protein-coupled membrane receptor type 5. RESULTS: In the cohort, 52% (21/40) of children had abnormal LV geometry; the optimal bile acid concentration to detect this abnormality with 70% sensitivity and 64% specificity was 152 µmol/L (C-statistics=0.68). Children with bile acid concentrations >152 µmol/L had ∼8-fold increased odds of detecting abnormalities in LVM, LVM index, left atrial volume index, and LV internal diameter. Serum bile acids positively correlated with LVM, LVM index, and LV internal diameter. Separately, Takeda G-protein-coupled membrane receptor type 5 protein was detected in myocardial vasculature and cardiomyocytes on immunohistochemistry. CONCLUSION: This association highlights the unique role of bile acids as one of the targetable potential triggers for myocardial structural changes in BA.

Application of organ dysfunction assessment scores following pediatric lung transplantation.

OBJECTIVE: Organ dysfunction (OD) after lung transplantation can reflect preoperative organ failure, intraoperative acute organ damage and post-operative complications. We assessed two OD scoring systems, both the PEdiatric Logistic Organ Dysfunction (PELOD) and the pediatric Sequential Organ Failure Assessment (pSOFA) scores, in recognizing risk factors for morbidity as well as recipients with prolonged post-transplant morbidity. DESIGN: Medical records of recipients from January 2009 to March 2016 were reviewed. PELOD and pSOFA scores were calculated on post-transplant days 1-3. Risk factors assessed included cystic fibrosis (CF), prolonged surgical time and worst primary graft dysfunction (PGD) score amongst others. Patients were classified into three groups based on their initial scores (group A) and subsequent trends either uptrending (group B) or downtrending (group C). Morbidity outcomes were compared between these groups. RESULTS: Total 98 patients were enrolled aged 0-20 years. Risk factors for higher pSOFA scores ≥ 5 on day 1 included non-CF diagnosis and worst PGD scores (p = .0006 and p = .03, respectively). Kruskal Wallis analysis comparing pSOFA group A versus B versus C scores showed significantly prolonged ventilatory days (median 1 vs. 4 vs. 2, p = .0028) and ICU days (median 4 vs. 10 vs. 6, p = .007). Similarly, PELOD group A versus B versus C scores showed significantly prolonged ventilatory days (1 vs. 5 vs. 2, p = < .0001). CONCLUSION: Implementing pSOFA scores bedside is a more effective tool compared to PELOD in identifying risk factors for worsened OD post-lung transplant and can be valuable in providing direction on morbidity outcomes in the ICU.

A novel interaction between extracellular vimentin and fibrinogen in fibrin formation.

INTRODUCTION: Thrombosis is frequently manifested in critically ill patients with systemic inflammation, including sepsis and COVID-19. The coagulopathy in systemic inflammation is often associated with increased levels of fibrinogen and D-dimer. Because elevated levels of vimentin have been detected in sepsis, we sought to investigate the relationship between vimentin and the increased fibrin formation potential observed in these patients. MATERIALS AND METHODS: This hypothesis was examined by using recombinant human vimentin, anti-vimentin antibodies, plasma derived from healthy and critically ill patients, confocal microscopy, co-immunoprecipitation assays, and size exclusion chromatography. RESULTS: The level of vimentin in plasma derived from critically ill subjects with systemic inflammation was on average two-fold higher than that of healthy volunteers. We determined that vimentin directly interacts with fibrinogen and enhances fibrin formation. Anti-vimentin antibody effectively blocked fibrin formation ex vivo and caused changes in the fibrin structure in plasma. Additionally, confocal imaging demonstrated plasma vimentin enmeshed in the fibrin fibrils. Size exclusion chromatography column and co-immunoprecipitation assays demonstrated a direct interaction between extracellular vimentin and fibrinogen in plasma from critically ill patients but not in healthy plasma. CONCLUSIONS: The results describe that extracellular vimentin engages fibrinogen in fibrin formation. In addition, the data suggest that elevated levels of an apparent aberrant extracellular vimentin potentiate fibrin clot formation in critically ill patients with systemic inflammation; consistent with the notion that plasma vimentin contributes to the pathogenesis of thrombosis.

Intravital Microscopy to Study Platelet-Leukocyte-Endothelial Interactions in the Mouse Liver.

Inflammation and thrombosis are complex processes that occur primarily in the microcirculation. Although standard histology may provide insight into the end pathway for both inflammation and thrombosis, it is not capable of showing the temporal changes that occur throughout the time course of these processes. Intravital microscopy (IVM) is the use of live-animal imaging to gain temporal insight into physiologic processes in vivo. This method is particularly powerful when assessing cellular and protein interactions within the circulation due to the rapid and sequential events that are often necessary for these interactions to occur. While IVM is an extremely powerful imaging methodology capable of viewing complex processes in vivo, there are a number of methodological factors that are important to consider when planning an IVM study. This paper outlines the process of conducting intravital imaging of the liver, identifying important considerations and potential pitfalls that may arise. Thus, this paper describes the use of IVM to study platelet-leukocyte-endothelial interactions in liver sinusoids to study the relative contributions of each in different models of acute liver injury.

Histone-stimulated platelet adhesion to mouse cremaster venules in vivo is dependent on von Willebrand factor.

OBJECTIVE: Extracellular histones are known mediators of platelet activation, inflammation, and thrombosis. Von Willebrand Factor (vWF) and Toll-like receptor 4 (TLR4) have been implicated in pro-inflammatory and prothrombotic histone responses. The objective of this study was to assess the role of vWF and TLR4 on histone-induced platelet adhesion in vivo. METHODS: Intravital microscopy of the mouse cremaster microcirculation, in the presence of extracellular histones or saline control, was conducted in wild-type, vWF-deficient, and TLR4-deficient mice to assess histone-mediated platelet adhesion. Platelet counts following extracellular histone exposure were conducted. Platelets were isolated from vWF-deficient mice and littermates to assess the role of vWF on histone-induced platelet aggregation. RESULTS: Histones promoted platelet adhesion to cremaster venules in vivo in wild-type animals, as well as in TLR4-deficient mice to a comparable degree. Histones did not lead to increased platelet adhesion in vWF-deficient mice, in contrast to littermate controls. In all genotypes, histones resulted in thrombocytopenia. Histone-induced platelet aggregation ex vivo was similar in vWF-deficient mice and littermate controls. CONCLUSIONS: Histone-induced platelet adhesion to microvessels in vivo is vWF-dependent and TLR4-independent. Platelet-derived vWF was not necessary for histone-induced platelet aggregation ex vivo. These data are consistent with the notion that endothelial vWF, rather than platelet vWF, mediates histone-induced platelet adhesion in vivo.

A high-throughput microfluidic device based on controlled incremental filtration to enable centrifugation-free, low extracorporeal volume leukapheresis.

Leukapheresis, the extracorporeal separation of white blood cells (WBCs) from red blood cells (RBCs) and platelets (PLTs), is a life-saving procedure used for treating patients with cancer and other conditions, and as the initial step in the manufacturing of cellular and gene-based therapies. Well-tolerated by adults, leukapheresis poses a significant risk to neonates and low-weight infants because the extracorporeal volume (ECV) of standard centrifugation-based machines represents a particularly large fraction of these patients' total blood volume. Here we describe a novel high-throughput microfluidic device (with a void volume of 0.4 mL) based on controlled incremental filtration (CIF) technology that could replace centrifugation for performing leukapheresis. The CIF device was tested extensively using whole blood from healthy volunteers at multiple hematocrits (5-30%) and flow rates (10-30 mL/min). In the flow-through regime, the CIF device separated WBCs with > 85% efficiency and 10-15% loss of RBCs and PLTs while processing whole blood diluted with saline to 10% hematocrit at a flow rate of 10 mL/min. In the recirculation regime, the CIF device demonstrated a similar level of separation performance, virtually depleting WBCs in the recirculating blood (~ 98% reduction) by the end of a 3.5-hour simulated leukapheresis procedure. Importantly, the device operated without clogging or decline in separation performance, with minimal activation of WBCs and PLTs and no measurable damage to RBCs. Compared to the typical parameters of centrifugation-based leukapheresis, the CIF device had a void volume at least 100-fold smaller, removed WBCs about twice as fast, and lost ~ 2-3-fold fewer PLTs, while operating at a flow rate compatible with the current practice. The hematocrit and flow rate at which the CIF device operated were significantly higher than previously published for other microfluidic cell separation methods. Finally, this study is the first to demonstrate a highly efficient separation of cells from recirculating blood using a microfluidic device. Overall, these findings suggest the feasibility of using high-throughput microfluidic cell separation technology to ultimately enable centrifugation-free, low-ECV leukapheresis. Such a capability would be particularly useful in young children, a vulnerable group of patients who are currently underserved.

High levels of von Willebrand factor with reduced specific activities in hospitalized patients with or without COVID-19.

The COVID-19 pandemic is often accompanied by severe respiratory illness and thrombotic complications. Von Willebrand Factor (VWF) levels are highly elevated in this condition. However, limited data are available on the qualitative activity of VWF in COVID-19. We measured plasma VWF levels quantitatively (VWF antigen) and qualitatively (ristocetin-induced platelet agglutination, glycoprotein IbM (GPIbM) binding, and collagen binding). Consistent with prior reports, VWF antigen levels were significantly elevated in hospitalized patients with or without COVID-19. The GPIbM and collagen binding activity-to-antigen ratios were significantly reduced, consistent with qualitative changes in VWF in COVID-19. Of note, critically ill hospitalized patients without COVID-19 had similar reductions in VWF activity-to-antigen ratios as patients with COVID-19. Our data suggest that qualitative changes in VWF in COVID-19 may not be specific to COVID-19. Future studies are warranted to determine the mechanisms responsible for qualitative changes in VWF in COVID-19 and other critical illnesses.• VWF levels were increased in COVID-19 compared to healthy controls.• VWF activity-to-antigen ratios were decreased in COVID-19 compared to healthy controls.• There were no differences in VWF activity-to-antigen ratios between hospitalized patients with or without COVID-19.• These findings are consistent with qualitative changes in VWF in systemic inflammation which are not specific to COVID-19.• Future studies are needed to define possible roles of changes in conformation or multimer length in the qualitative changes in VWF in systemic inflammation.

Aberrant Fibrin Clot Structure Visualized Ex Vivo in Critically Ill Patients With Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

OBJECTIVES: Disseminated fibrin-rich microthrombi have been reported in patients who died from COVID-19. Our objective is to determine whether the fibrin clot structure and function differ between critically ill patients with or without COVID-19 and to correlate the structure with clinical coagulation biomarkers. DESIGN: A cross-sectional observational study. Platelet poor plasma was used to analyze fibrin clot structure; the functional implications were determined by quantifying clot turbidity and porosity. SETTING: ICU at an academic medical center and an academic laboratory. PATIENTS: Patients admitted from July 1 to August 1, 2020, to the ICU with severe acute respiratory syndrome coronavirus 2 infection confirmed by reverse transcription-polymerase chain reaction or patients admitted to the ICU with sepsis. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Blood was collected from 36 patients including 26 ICU patients with COVID-19 and 10 ICU patients with sepsis but without COVID-19 at a median of 11 days after ICU admission (interquartile range, 3-16). The cohorts were similar in age, gender, body mass index, comorbidities, Sequential Organ Failure Assessment (SOFA) score, and mortality. More patients with COVID-19 (100% vs 70%; p = 0.003) required anticoagulation. Ex vivo fibrin clots formed from patients with COVID-19 appeared to be denser and to have smaller pores than those from patients with sepsis but without COVID-19 (percent area of fluorescent fibrin 48.1% [SD, 16%] vs 24.9% [SD, 18.8%]; p = 0.049). The turbidity and flow-through assays corroborated these data; fibrin clots had a higher maximum turbidity in patients with COVID-19 compared with patients without COVID-19 (0.168 vs 0.089 OD units; p = 0.003), and it took longer for buffer to flow through these clots (216 vs 103 min; p = 0.003). In patients with COVID-19, d-dimer levels were positively correlated with percent area of fluorescent fibrin (ρ = 0.714, p = 0.047). Denser clots (assessed by turbidity and thromboelastography) and higher SOFA scores were independently associated with delayed clot lysis. CONCLUSIONS: We found aberrant fibrin clot structure and function in critically ill patients with COVID-19. These findings may contribute to the poor outcomes observed in COVID-19 patients with widespread fibrin deposition.